HNSCC Tumor-Derived Organoid Generation

HNSCC patient tumor derived organoids were generated as previously described with some modifications^{45 (link)}. Briefly, tumors were first cut and minces into small pieces, which were then incubated in a digestion medium (including 1 mg/mL Collagenase XI, 10 μg/mL DNAase I, 10.5 μmol/L Y-27632 in human complete medium) at 37 °C for 30 min, the supernatant was collected and filtered through 70 μm filters and centrifugated at 800 rpm for 5 min. The pellet was then resuspended in Matrigel matrix (Corning) and seeded onto a 24-well plate for 15 min inside a CO₂ incubator at 37 °C. Finally, each well of the 24-well plate was incubated with 1 mL of tumor organoid culturing medium, which included advanced DMEM/F12 medium containing HEPES 10 mmol/L, 1X Glutmax, A83–01 500 nmol/L, hEGF 50 ng/mL, mNoggin 100 ng/mL, hFGF10 100 ng/mL, hGastrin I 0.01 μmol/L, nicotinamide 10 mmol/L, N-acetylcysteine 1.25 mmol/L, PGE2 1 μmol/L, B27 supplement, R-spondin1 conditioned media, and Afamin/Wnt3A conditioned media. For co-culture experiment, sCAFs or rCAFs were first seeded onto a 24-well plate overnight, while organoids mixed with Matrigel matrix (Corning) were then placed on top of these cells in the presence or absence of DDP -/+ cetuximab.